ABSTRACT In this competing renewal of AR057025, we expand the scope and now focus on collagen in nucleus pulposus (NP) tissue of the interertebral disc. We propose to 1) define the role of NP-specific post- translational modifications within type II collagen chains in regulating the diameter of fibrils. 2) establish a molecular fingerprint for cross-linked collagen heterofibril assembly as a biomarker for native and in vitro generated NP tissue. By the age of fifty, 85 percent of the US population shows evidence of a compromised collagen network and disc herniation. As the population ages, such biomarkers to evaluate the quality of regenerated NP neo-tissue is significant, offering new hope in the treatment of disc disease. The fibrillar network that frames the jelly-like nucleus pulposus is made up of types II, IX and XI collagen, the same gene products that characterize hyaline cartilage. The mechanism that drives these molecules to heteropolymerize as thin diameter fibrils in nucleus pulposus but as thicker diameter fibrils in hyaline cartilage is still unclear. Recent evidence convincingly correlates the assembly of thin (<20nm) collagen fibrils in NP with elevated levels of an unique type II collagen post-translational modification, the 3-hydroxylation of proline residue 944 (P944). In hyaline cartilage where the 3-hydroxylation of P944 is nearly lacking, thicker (20- 100nm) fibrils are observed. We will use the RCS-LTC cell line as a model system to address this mechanism. This cell line, originally derived from a spinal neoplasm, assembles types II, IX, XI collagens into cross- linked thin diameter collagen fibrils in a jelly-like extracellular matrix. Elevated levels of the prolyl 3-hydroxylase 2 (P3H2) enzyme correlated with the highly 3-hydroxylated P944 residues in type II collagen chains deposited in the matrix. We will use the CRISPR/Cas9 gene editing system to knock out the P3H2 gene, in combination with mass spectrometry and electron microscopy to define a role for 3-hydroxyproline residues in type II collagen fibril diameter regulation. We intend to aggressively pursue this concept in order to understand how cells modulate the thickness of collagen fibrils in NP and other type II collagen based tissues. This is important from both a basic biology and tissue regeneration perspective. Furthermore, employing biochemical methodology generated from our original RO1 grant, we aim to fingerprint the pattern of collagen inter-type II- IX-XI cross-linking in native and in vitro cultured nucleus pulposus neo-tissue. This will provide a screen for normal matrix assembly and serve as a basis for future regeneration studies. Electron microscopy, mass spectrometry and biomechanics will be used to determine the thickness and post-translational quality of the fibrils and function. The goal is to ascertain the ability of the neo-tissues to assemble tissue-specific 3- hydroxyproline modified type II collagen molecules into a network typical of native nucleus pulposus. Essential information for next-generation NP neo-tissue with high collagen content will be gained. This is significant as a nascent, accurately cross-linked, type II-IX-XI collagen template is crucial to the growth of the fibril.